molecules

Communication
Development and Validation of a HPLC-UV
Method for Extraction Optimization and Biological
Evaluation of Hot-Water and Ethanolic Extracts of
Dendropanax morbifera Leaves
Hyung-Jae Choi 1,† , Dae-Hun Park 2,† , Seung-Hui Song 1 , In-Soo Yoon 3, * ID
and
Seung-Sik Cho 1, * ID
1 Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National
University, Muan, Jeonnam 58554, Korea; qweert15@naver.com (H.-J.C.); tmdgml7898@naver.com (S.-H.S.)
2 Department of Nursing, Dongshin University, Naju, Jeonnam 58245, Korea; dhj1221@hanmail.net
3 Department of Manufacturing Pharmacy, College of Pharmacy, Pusan National University, Geumjeong,
Busan 46241, Korea
* Correspondence: insoo.yoon@pusan.ac.kr (I.-S.Y.); sscho@mokpo.ac.kr (S.-S.C.);
Tel.: +82-51-510-2806 (I.-S.Y.); +82-61-450-2687 (S.-S.C.)
† These authors contributed equally to this work.

Received: 22 February 2018; Accepted: 12 March 2018; Published: 13 March 2018

Abstract: Dendropanax morbifera Leveille (Araliaceae) has been used in traditional oriental remedies
for cancer, inflammation, diabetes, and thrombosis. However, a validated analytical method,
standardization, and optimization of extraction conditions with respect to biological activity have
not been reported. In this study, a simple and validated HPLC method for identifying and
quantifying active substances in D. morbifera was developed. Hot water and ethanolic D. morbifera
leaf extracts from different production regions were prepared and evaluated with regard to their
chemical compositions and biological activities. The contents of active compounds such as rutin and
chlorogenic acid were determined in four samples collected from different regions. The 80% ethanolic
extract showed the best antioxidant activity, phenolic content, reducing power, and xanthine oxidase
(XO) inhibitory activity. The validated HPLC method confirmed the presence of chlorogenic acid and
rutin in D. morbifera leaf extracts. The antioxidant and XO inhibitory activity of D. morbifera extract
could be attributed to the marker compounds. Collectively, these results suggest that D. morbifera
leaves could be beneficial for the treatment or prevention of hyperuricemia-related disease, and the
validated HPLC method could be a useful tool for the quality control of food or drug formulations
containing D. morbifera.

Keywords: Dendropanax morbifera; extraction optimization; HPLC; xanthine oxidase

1. Introduction
Dendropanax morbifera Leveille is a subtropical evergreen tree which belongs to the family
Araliacea, and it has been used in traditional medicines for the treatment of infectious diseases,
dermatopathy, and headaches [1]. Previous studies have reported that D. morbifera exhibit
various pharmacological effects, including antioxidant [2], antidiabetic [3], hepatoprotective [4],
anticomplementary [5], and antiatherogenic activities [6]. D. morbifera leaf has also been traditionally
used as a botanical remedy in Asia [7]. The efficacy of extracts and active constituents prepared
with D. morbifera leaves as a medicinal source has been investigated in a few studies to date. Several
research studies have reported various pharmacological activities of D. morbifera leaves and their

Molecules 2018, 23, 650; doi:10.3390/molecules23030650 www.mdpi.com/journal/molecules

Molecules 2018, 23, 650 2 of 11

active ingredients identified, which are listed in Table 1. Recently there has been a great effort to find
candidates from natural products to effectively control metabolic disease [8].
D. morbifera leaves were reported to possess antioxidant and anticancer activities by moduating
cellular apoptosis in various human tumor cell lines such as colon adenocarcinoma cells, biliary
tract cells, hepatocellular carcinoma cells, and human osteocarcinoma cells [1]. Additionally,
anti-inflammatory, antithrombotic, and other activities were found in D. morbifera leaf extracts, as
shown in Table 1. However, the effects of D. morbifera leaf extracts on the activity of xanthine oxidase
(XO) have not been reported. In the previous reports listed in Table 1, D. morbifera leaf extracts
were prepared with organic solvents such as ethanol, methanol, and chloroform. However, there
have been few studies on the optimization of extraction conditions with respect to biological activity,
phytochemical contents, or both.

Table 1. Pharmacological activities and/or chemical constituents of D. morbifera leaf extracts reported
in previous literatures.

Ext. Solvent Activity Chemical Identified Region Ref.

chloroform anti-inflammatory - - [4]
70% ethanol anticancer - - [9]
- anti-inflammatory oleifoliside A - [10]
chloroform kidney damage - - [11]
- anticomplement three polyacetylenes - [5]
methanol anti-inflammatory rutin and 21 compounds Jeju, Korea [12]
antioxidant chlorogenic acid
80% ethanol Jeju, Korea [1]
anticancer caffeic acid, rutin, rosmarinic acid
- Antithrombotic rutin Wando, Korea [13]
- Anticancer oleifoliside B - [14]
- Neurogenerative rutin Wando, Korea [15]

Recently, considerable effort has been focused on developing D. morbifera leaf as a therapeutic
or functional source, but no positive results have been reported. To facilitate the pharmaceutical
industrialization of D. morbifera leaf, a standardization process of the plant material with marker
compounds identified using validated analytical methods is highly required. However, no validated
analytical methods for the standardization and optimization of the biological properties of D. morbifera
preparations have been reported in the literature. Here, we report the preparation of various hot water
and ethanolic extracts of D. morbifera leaves collected from four different regions in Korea as well as
the development of a simple and validated high-performance liquid chromatography (HPLC) method
for chemical profiling and standardization of the plant extracts. In addition, the extraction process was
optimized for phenolic contents and biological activities such as antioxidant and XO inhibitory activity.

2. Results and Discussion

2.1. Optimization of Chromatographic Conditions

The HPLC conditions were optimized for the mobile phase composition, column temperature,
wavelength, and flow rate (data not shown). A gradient program was used to separate the active
markers in a single run within a reasonable period. Detection wavelengths were set according to the
ultraviolet (UV) absorption maxima of the compounds (330 nm). The chemical structures of the active
constituents identified and representative chromatograms of the standard mixture and sample extracts
are shown in Figure 1.
Molecules 2018,
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Figure 1. Chromatogram
Figure of standard
1. Chromatogram of standard D. D.
andand morbifera leafleaf
morbifera extract.
extract.

2.2.2.2.
Method Validation
Method Validation
TheThe
limit of detection
limit of detection(LOD)
(LOD)of an analytical
of an procedure
analytical procedureis the lowest
is the lowest amount
amount of an analyte
of an in ain a
analyte
sample thatthat
sample can can
be detected but not
be detected butnecessarily quantified
not necessarily [16] while
quantified [16]the limitthe
while of quantification (LOQ)
limit of quantification
is the lowest
(LOQ) level
is the in thelevel
lowest linear
inconcentration range with acceptable
the linear concentration range withprecision
acceptable and accuracy.
precision Theaccuracy.
and LOD
of the
Thepresent
LOD method was 0.65 method
of the present and 0.50 was
µg/mL forand
0.65 chlorogenic
0.50 μg/mLacid and
for rutin, respectively
chlorogenic acid (Table 2),
and rutin,
andrespectively
the corresponding
(Table 2),LOQandvalues were 2.13 and
the corresponding 1.64values
LOQ µg/mL, wererespectively
2.13 and 1.64(Table 2). Calibration
μg/mL, respectively
curves were
(Table 2). linear over a curves
Calibration concentration rangeover
were linear of 6.25–50 µg/mL forrange
a concentration the two markers.μg/mL
of 6.25–50 The calibration
for the two
2
curves exhibited
markers. good linearcurves
The calibration regressions (coefficient
exhibited good linear regressions r(coefficient
of determination > 0.999 for ofchlorogenic
determination acidr2 >
and0.999
rutin,
forTable 2).
chlorogenic acid and rutin, Table 2).

Table 2. HPLC
Table data
2. HPLC of calibration
data graphs
of calibration andand
graphs limit of quantification
limit andand
of quantification detection of two
detection markers.
of two markers.

Analyte Retention Time Retention

Analyte (min) Time
r2 Range
r2
Range
(µg/mL) LOQ
LOQ (µg/mL) LOD
LOD (µg/mL)
(min) (μg/mL) (μg/mL) (μg/mL)
Chlorogenic acid 7.7 0.9988 3.125–50 2.13 0.65
Rutin Chlorogenic acid
12.7 7.7 0.9996 0.9988 3.125–50
3.125–50 2.13
1.64 0.65 0.50
Rutin 12.7 0.9996 3.125–50 1.64 0.50

The results of the intraday and interday precision experiments are shown in Table 3.
The results of the intraday and interday precision experiments are shown in Table 3. The
The developed method was precise, as indicated by the relative standard deviation (RSD) values
developed method was precise, as indicated by the relative standard deviation (RSD) values (less
(less than 2.5%) for the repeatability of the intraday and interday precision studies, which were below
than 2.5%) for the repeatability of the intraday and interday precision studies, which were below
the limit recommended by the International Conference on Harmonisation (ICH) guidelines [17].
the limit recommended by the International Conference on Harmonisation (ICH) guidelines [17].
The overall recovery percentages were in the range of 92.36–98.12% for chlorogenic acid and
The overall recovery percentages were in the range of 92.36–98.12% for chlorogenic acid and
98.36–106.83% for rutin. These results demonstrate that the developed method was reproducible
98.36–106.83% for rutin. These results demonstrate that the developed method was reproducible
with good accuracy (Table 3).
with good accuracy (Table 3).
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Table 3. Analytical results of intra-day and inter-day precision and accuracy.

Table 3. Analytical results of intra-day and inter-day precision and accuracy.
Conc. Intra-Day (n = 3) Inter-Day (n = 3)
Analyte Intra-Day
a (n = 3) (%) RSD (%) Inter-Day (n = 3)
Analyte
(μg/mL)
Conc. (µg/mL) RSD (%) Accuracy Accuracy (%)
12.5 RSD
4.73 (%) Accuracy
92.36 (%) RSD
1.17 (%) Accuracy
94.78 (%)
Chlorogenic acid 12.525 4.73
1.11 92.36
95.70 1.641.17 97.0794.78
Chlorogenic acid 2550 1.11 95.70
2.12 96.12 1.951.64 98.1297.07
50 2.12 96.12 1.95 98.12
12.5 3.18 100.65 7.15 106.83
12.5 3.18 100.65 7.15 106.83
Rutin 25 1.00 99.07 5.71 102.17
Rutin 25 1.00 99.07 5.71 102.17
5050 1.19
1.19 98.36
98.36 1.831.83 100.60
100.60

The results of the repeatability experiments are shown in Table 4. The developed method was
The results of the repeatability experiments are shown in Table 4. The developed method was
precise; the RSD values for the repeatability precision studies were below 2.0%.
precise; the RSD values for the repeatability precision studies were below 2.0%.
Table 4. Analytical recovery data (n = 6).
Table 4. Analytical recovery data (n = 6).
Analyte Added (μg/mL) Recovery (%) (Mean ± SD) RSD (%)
Analyte Added 12.5
(µg/mL) Recovery95.77 ± 0.62 ± SD)
(%) (Mean RSD
0.78 (%)
Chlorogenic acid 12.5
25 96.72±± 0.62
95.77 1.33 0.78
1.50
Chlorogenic acid 25 96.72 ± 1.33 1.50
50 98.21 ± 0.45 0.48
50 98.21 ± 0.45 0.48
12.5 102.73 ± 0.40 0.40
12.5 102.73 ± 0.40 0.40
RutinRutin 2525 97.65±± 1.49
97.65 1.49 1.56
1.56
5050 98.64±± 0.43
98.64 0.43 0.43
0.43

2.3.Contents
2.3. ContentsofofMarker
MarkerCompounds
Compoundsin
inD.
D.morbifera
morbiferafrom
fromDifferent
DifferentCultivation
CultivationRegions
Regions
Plantsamples
Plant sampleswere
werecollected
collectedfrom
fromfour
fourdifferent
differentcultivation
cultivationregions
regionstotocompare
comparethe theextraction
extraction
yield and productivity of the active substances. The extraction yield and content
yield and productivity of the active substances. The extraction yield and content of markers were of markers were
compared for
compared forthe
thevarious
varioushothotwater
waterextracts
extractsofofthe
theplant.
plant.The
Thevalidated
validatedHPLC
HPLCmethod
methodwas wasused
usedtoto
analyze the four different extracts. Chlorogenic acid and rutin were commonly identified
analyze the four different extracts. Chlorogenic acid and rutin were commonly identified in all samples. in all
samples.
The The extraction
extraction yield of
yield of samples samples
from from the
the various various
regions regionsindecreased
decreased in theorder:
the following following order:
Jangheung
Jangheung
(JH, 13.46%)(JH, 13.46%)
> Wando (W,>12.03%)
Wando >(W, 12.03%)
Kangjin (K,>11.0%)
Kangjin (K, (JJ,
> Jeju 11.0%) > Jeju
8.2%), (JJ, 8.2%),
indicating thatindicating that
the JJ sample
the JJ sample showed the lowest yield. The average amounts (wt %) of chlorogenic
showed the lowest yield. The average amounts (wt %) of chlorogenic acid and rutin are presented in acid and rutin
are presented
Figure 2. in Figure 2.

1.8 3.0

1.6 *
2.5
*
Chlorogenic acid (%, w/w)

1.4

2.0
Rutin (%, w/w)

1.2

1.0 * 1.5
0.8 *
0.6 1.0

0.4
* 0.5
0.2
*
0.0 0.0
W K JH JJ W K JH JJ

Figure 2. Contents of chlorogenic acid and rutin in hot water extracts of D. morbifera leaf from different
Figure 2. Contents of chlorogenic acid and rutin in hot water extracts of D. morbifera leaf from different
cultivation regions. W: Wando, K: Kangjin, JH: Jangheung, JJ: Jeju. The asterisk represents a value
cultivation regions. W: Wando, K: Kangjin, JH: Jangheung, JJ: Jeju. The asterisk represents a value
significantly different from the other groups (p < 0.05). Values are mean ± standard deviation (n = 3).
significantly different from the other groups (p < 0.05). Values are mean ± standard deviation (n = 3).
The contents of chlorogenic acid were highest in the W sample, while the contents of rutin were
The
highest in contents of chlorogenic
the K sample. The sum ofacid wereactive
the two highest in the W
ingredient sample,
contents while the acid
(chlorogenic contents of rutin
and rutin) of
W and K was comparable to each other, whereas the extraction yield of W was higher than that ofand
were highest in the K sample. The sum of the two active ingredient contents (chlorogenic acid K.
rutin) of W and K was comparable to each other, whereas the
Thus, D. morbifera from region W was used for further experiments. extraction yield of W was higher than
that of K. Thus, D. morbifera from region W was used for further experiments.
Molecules 2018, 23, 650 5 of 11

Molecules 2018, 23, x FOR PEER REVIEW 5 of 11

Molecules 2018, 23, x FOR PEER REVIEW 5 of 11
2.4. Contents of Marker Compounds in D. morbifera Leaf Extracts
2.4. Contents of Marker Compounds in D. morbifera Leaf Extracts
2.4.
SixContents
extracts,of hot
Marker Compounds
water in D. morbifera
and ethanolic extractLeaf
with Extracts
varying ethanol contents (20 to 100%, v/v)
Six extracts, hot water and ethanolic extract with varying ethanol contents (20 to 100%, v/v)
were prepared and compared with respect to the marker contets using the validated HPLC method.
were Six extracts,
prepared andhot water and
compared withethanolic
respect to extract with varying
the marker ethanol
contets using thecontents
validated (20 to 100%,
HPLC v/v)
method.
Thewere
average contents
prepared and (wt %) of both
compared with markerstoare
respect the presented
marker in Figure
contets using 3,the
and their levels
validated HPLC inmethod.
the 80%
The average contents (wt%) of both markers are presented in Figure 3, and their levels in the 80%
ethanolic extract
The average
ethanolic were
extract greater
contents
were (wt%)than
greater those
ofthan
both in the
markers
those in other ethanolic
are other
the presented extracts
in Figure
ethanolic (chlorogenic
3, and
extracts their acid: 4.71
levelsacid:
(chlorogenic ±4.71
in the 0.06%;
80%±
rutin: 3.29 ±
0.06%; rutin: 3.29 ± 0.04%). Thus, 80% ethanol was selected as the most efficient extraction solvent. ±
ethanolic extract
0.04%). were
Thus, greater
80% than
ethanol those
was in the
selected other
as ethanolic
the most extracts
efficient (chlorogenic
extraction acid:
solvent. 4.71
0.06%; rutin: 3.29 ± 0.04%). Thus, 80% ethanol was selected as the most efficient extraction solvent.
6 4
6 4
* *
5
*
w/w)

5 * 3
w/w)

* 3
(%,(%,

4
*

w/w)
4
*

w/w)
acid

(%,(%,
*
acid

3 2
*
Chlorogenic

3 2

Rutin
*
Chlorogenic

Rutin
2
2 1
1 1
1

0 0
0 0% 20% 40% 60% 80% 100% 0 0% 20% 40% 60% 80% 100%
0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%
Figure 3. Contents of chlorogenic acid and rutin in hot water (0%) and ethanolic extracts (20–100%)
Figure
of D. 3. Contents
morbifera of chlorogenic
leaf. Ethanolic acidwere
extracts and prepared
rutin in hot water
with (0%) and
20–100% ethanolic
ethanol. Theextracts
asterisk(20‒100%)
represents
Figure
of D. 3. Contents
morbifera leaf. of chlorogenic
Ethanolic acidwere
extracts and rutin in hot
prepared water
with (0%) and
20‒100% ethanolic
ethanol. The extractsrepresents
asterisk (20‒100%)
a valueD.significantly different from the other groups (pwith
< 0.05). Values mean ± standard
were theasterisk
morbifera
aofvalue leaf.
significantly Ethanolic extracts
different from thewere
otherprepared 20‒100%
groups (p < 0.05). ethanol.
Values wereThe
the mean ± represents
standard
deviation (n = 3).
a value significantly different from the other groups (p < 0.05). Values were the mean ± standard
deviation (n = 3).
deviation (n = 3).
2.5.2.5.
Antioxidant
Antioxidant Activity
Activityand
andTotal
TotalPhenolic
PhenolicContents
ContentsofofD. D.morbifera
morbiferaExtracts
Extracts
2.5. Antioxidant Activity and Total Phenolic Contents of D. morbifera Extracts
TheTheantioxidant
antioxidant potential
potentialof of
various
various ethanoilc
ethanoilc extracts
extracts D.D.
ofof morbifera
morbifera was determined
was determined using
usingthe
The antioxidant
2,2-diphenyl-1-picrylhydrazyl potential of
(DPPH) various ethanoilc
scavenging and extracts
reducing of D.
power
the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and reducing power assays. The DPPH morbifera
assays. was
The determined
DPPH using
scavenging
assaytheis 2,2-diphenyl-1-picrylhydrazyl
a fast assay
scavenging and easy method
is a fast and easy (DPPH)forscavenging
for evaluating
method the free radical
evaluating andfree
the reducing
scavenging power assays.
ability
radical scavenging The
ofability
given DPPH
ofsamples.
given
scavenging
Thesamples.
antioxidant assay
The effects is a fast
of plant
antioxidant and easy
extracts
effects method for
are generally
of plant evaluating
extracts are related the free radical
to therelated
generally phenolic scavenging
contents,
to the phenolicandability of given
phenolic-rich
contents, and
samples.
sources The antioxidant
of phytochemicals
phenolic-rich sources of with effects of plant
antioxidant with
phytochemicals extracts
activity are generally
have curative
antioxidant related
benefits
activity to the phenolic
have against contents,
curativeconditions andas
such
benefits against
phenolic-rich
inflammation,
conditions such sources
oxidative of phytochemicals
stress, and oxidative
as inflammation, with
other metabolic antioxidant
stress, anddiseases activity
other [17]. have curative
metabolic diseases [17]. benefits against
conditions such as inflammation, oxidative stress, and other metabolic diseasescorrelated
[17].
TheThe antioxidative
antioxidative properties
properties of theoftest
theplant
test extracts
plant extracts werecorrelated
were closely closely with the
with the composition
The
composition antioxidative
of active properties
compounds of the
suchTherefore,test
as phenolics. plant extracts
Therefore, the were closely
we phenolic correlated
comparedcontents
the phenolic with
contentsthe
of active compounds such as phenolics. we compared (mg/g as gallic
composition
(mg/g asvarious of active
gallic acid) compounds
of the various such as phenolics.
D. morbifera Therefore,
leaf extracts. we compared
The DPPH radical the phenolic contents
acid) of the D. morbifera leaf extracts. The DPPH radical scavenging activityscavenging
is shown inactivity
Figure 4.
(mg/g as gallic acid)4.ofThe
the various D. morbifera leafshowed
extracts. The DPPHDPPH radicalradical
scavenging activity
The 80% ethanolic extract showed the highest DPPH radical scavenging activity (36.10 ±scavenging
is shown in Figure 80% ethanolic extract the highest 2.68%) with
is shown
activity in Figure
(36.10 ± 2.68%) 4. with
The a80% ethanolic inhibitory
half-maximal extract showed the highest (IC50)DPPH radical scavenging
a half-maximal inhibitory concentration (IC50 ) of 116.97concentration
µg/mL. of 116.97 μg/mL.
activity (36.10 ± 2.68%) with a half-maximal inhibitory concentration (IC50) of 116.97 μg/mL.
50
50

40 *
*
(%)(%)

40
activity
activity

30
30
Antioxidant
Antioxidant

20
20

10
10

0
0 0% 20% 40% 60% 80% 100%
0% 20% 40% 60% 80% 100%

Figure 4. DPPH
Figure 4. DPPHscavenging
scavengingactivity
activityofofethanolic
ethanolicextracts
extracts of D. morbifera
of D. morbifera leaf.
leaf. Ethanolic
Ethanolicextracts
extractswere
were
Figure
prepared 4.
withDPPH scavenging
20–100% ethanol.activity
The of ethanolic
asterisk extracts
represents a of D.
value morbifera leaf.
significantly Ethanolic
different
prepared with 20‒100% ethanol. The asterisk represents a value significantly different from the extracts
from the were
other
prepared
groups
other with
(pgroups
< 0.05). <20‒100%
(p Values ethanol.
0.05). were the
Values The
mean
were asterisk
the±mean
standardrepresents
deviation
± standard a (n
value (nsignificantly
= 3).
deviation = 3). different from the
other groups (p < 0.05). Values were the mean ± standard deviation (n = 3).
Molecules 2018, 23, 650 6 of 11

The reducing power assay is also very useful for evaluating the antioxidant activity. In the present
study, we tested the reductive capability of extract sample by measuring the reduction of Fe3+ . The 80%
ethanolic extract exhibited the highest activity among the six extracts (Table 5). The reductive activity
expressed as vitamin C equivalents was 17.70 ± 0.40 µg/100 µg. The total phenolic content was
determined using the Folin–Ciocalteu method [17], and it was reported as gallic acid equivalents by
referencing the standard curve (r2 > 0.999), as shown in Table 5. The phenolic content of the 80%
ethanolic extract was higher than that of the other ethanolic extracts (57.89 ± 2.6 mg/g as gallic
acid equivalents). Taken together, the results indicate that the DPPH radical scavenging activity,
reducing power, and phenolic content were significantly higher in the 80% ethanolic extract than in
the other extracts.

Table 5. Reducing power and total phenolic contents of D. morbifera leaf extracts.

Reducing Power Total Phenolic Content

Extract
(Ascorbic Acid eq. µg/100 µg Extract) (Gallic Acid eq. mg/g)
Water 8.4 ± 0.2 30.27 ± 0.6
20% EtOH Ex 7.4 ± 0.7 26.3 ± 0.6
40% EtOH Ex 12.3 ± 0.5 39.28 ± 1.4
60% EtOH Ex 13.6 ± 0.2 52.30 ± 2.9
80% EtOH Ex 17.7 ± 0.4 57.89 ± 2.6
100% EtOH Ex 5.5 ± 1.3 34.72 ± 1.3

2.6. XO Inhibitory Activity of D. morbifera Extracts

The effect of ethanol concentration on the XO inhibitory activity of D. morbifera leaf ethanolic
extracts is shown in Figure 5. Allopurinol (ALP, positive control) at a concentration of 50 µg/mL
significantly inhibited XO activity (82.08 ± 0.82%). The XO inhibitory activity of the ethanolic extracts
was significantly higher in the 80% ethanolic extract than the other extracts. The XO inhibitory activity
of the 80% ethanolic extract tended to increase in a concentration-dependent manner with increasing the
extract concentration tested by up to 2 mg/mL (data not shown). Previously, we reported four different
botanical extracts as potential XO inhibitors [18]. Yoon et al. [19,20] reported that the optimized
extracts of Corylopsis coreana and Camellia japonica inhibited XO activity by approximately 50% at a
concentration of 2 mg/mL. Additionally, Yoon et al. [18] demonstrated that Quercus acuta extract
showed approximately 50% XO inhibitory activity at a concentration of 1 mg/mL. Cudrania tricuspidata
extract inhibited XO by approximately 75% at a concentration of 2 mg/mL [21]. The plant extracts with
XO inhibitory activity at 1 and 2 mg/mL demonstrated consistent effects in the in vivo animal disease
model. Thus, it is plausible that the 80% ethanolic extract of D. morbifera leaves could be developed as
a candidate antihyperuricemic agent.
We identified chlorogenic acid and rutin as marker compounds of extracts of D. morbifera leaves
from different cultivation regions. In the present study, we confirmed that these two compounds were
common constituents of the D. morbifera leaves from all four regions. This finding could be important in
the use of this plant for industrial purpose. Furthermore, we established the optimal analysis methods
and optimized the extraction conditions for further studies. Rutin is a flavonoid known to have
diverse pharmacological effects such as antihyperuricemia, anti-inflammatory, anticonvulsant, and
anti-Alzheimer’s disease [19,22–24]. A previous study reported that rutin at doses of 50 and 100 mg/kg
markedly reduced biological markers in hyperuricemic mice [23]. In another study, rutin exhibited
antihyperuricemic effects by inhibiting xanthine dehydrogenase/XO activity [24]. Chlorogenic acid has
also been reported to have diverse pharmacological effects such as anti-inflammatory, antiallergic, and
antihyperuricemic effects [25–28]. Meng et al. [27] reported that chlorogenic acid has anti-gout activity
by inhibiting XO activity. Chlorogenic acid also decreased the levels of proinflammatory cytokines
such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α induced by uric acid [27–29].
These results suggest that chlorogenic acid may have considerable potential for development as an
antihyperuricemic agent.
 Molecules 2018, 23, x FOR PEER REVIEW 7 of 11

Molecules 2018, 23, 650 7 of 11

results suggest that chlorogenic acid may have considerable potential for development as an
antihyperuricemic agent.
AsAsshown
shownininTable
Table1, 1, previous
previous studiesreported
studies reportedthe
thediverse
diverseactivities
activitiesofofextracts
extractsofofD.D.morbifera
morbifera
leaves. However,
leaves. However, to to the
the best
best of
of our
our knowledge,
knowledge, our our present
present study
study isisthe
thefirst
firsttotoreport
reportthethe
optimizationofofthe
optimization theextraction
extractionprocess
processofofpharmaceutically
pharmaceuticallyactive
activeindicators
indicatorsfrom
fromD.D.morbifera
morbifera leaves
leaves
from
from various
various regions
regions andand the comparation
the comparation of antioxidant
of antioxidant and XOand XO inhibitory
inhibitory activitiesactivities
of variousof region-
various
region-specific
specific extracts. extracts.

120

Xanthine oxidase activity (%)

100

80
*
60

40

20 *
0
CON ALP 0% 20% 40% 60% 80% 100%

Figure
Figure 5. 5. Xanthineoxidase
Xanthine oxidaseinhibitory
inhibitoryactivities
activitiesininethanolic
ethanolic(0–100%)
(0–100%)extracts
extractsofofD.D.morbifera
morbiferaleaves
leaves
(2 (2 mg/mL)
mg/mL) andand allopurinol
allopurinol (ALP,(ALP, 50 μg/mL).
50 µg/mL). EthanolicEthanolic
extractsextracts were prepared
were prepared with 20–100%with ethanol.
20‒100%
Theethanol.
asteriskThe asteriska represents
represents a valuedifferent
value significantly significantly different
from the from the
other groups (p <other
0.05).groups (p < 0.05).
Values were the
mean ± standard
Values were thedeviation
mean ± standard
(n = 3). deviation (n = 3).

AAstandard
standardanalytical
analyticalmethod
methodis iscrucial
crucialforforthe
theindustrial
industrialapplication
applicationofofplant
plantextracts,
extracts,andand
extraction optimization is an essential process for the optimization and quality
extraction optimization is an essential process for the optimization and quality control of natural control of natural
products
products from
from various
various sources.
sources. However,
However, there there
has beenhasno been no of
report report of a standard
a standard profile forprofile for D.
D. morbifera.
morbifera. Moreover, currently, no other validated HPLC method
Moreover, currently, no other validated HPLC method has been reported for the simultaneous has been reported for the
simultaneous determination of chlorogenic acid and rutin in the Dendropanax
determination of chlorogenic acid and rutin in the Dendropanax genus. In Korea, one species of genus. In Korea, one
D.species of D.
morbifera morbifera
is mainly is mainlyincultivated
cultivated in four
four different different
regions regions
studied (W, studied (W,JJ).
K, JH, and K,InJH, and
this JJ). Inthe
study, this
study, the differences in the marker content of samples from the four production
differences in the marker content of samples from the four production regions were compared using our regions were
compared
validated HPLCusing our validated
methods, which showedHPLCefficiency
methods, which
in the showed
analysis efficiency inofthe
and optimization the analysis
D. morbiferaand
optimization of the D. morbifera leaf preparations. Moreover, the findings of the present
leaf preparations. Moreover, the findings of the present study could be applied to the industrialization study could
ofbe
D.applied
morbiferatobythe industrialization
providing of D. morbifera
basic information on samplesby providing
from the basic information
four cultivation on samples
regions in Korea.from
the four cultivation regions in Korea.
3. Experimental Section
3. Experimental Section
3.1. Plant Material and Extract Preparation
3.1. Plant Material and Extract Preparation
D. morbifera leaves were collected in October 2016 near Wando (34.3110596 N, 126.755054 E),
Kangjin D. (34.642077
morbifera leaves were collected
N, 126.76726 E), andin Jangheung
October 2016 near Wando
(34.681685 (34.3110596N,
N, 126.906927 126.755054E),
E) in Jeonnam
Kangjin Korea
Province, (34.642077N,
and Jeju126.76726E),
(33.499621 and JangheungE)
N, 126.531188 (34.681685N, 126.906927E)
in Jeju Province, Korea. in Jeonnam
Voucher Province,
specimens
Korea and Jeju (33.499621N,
(MNUCSS-DMWD-01, MNUCSS-DMJH-01, 126.531188E) in Jeju Province,andKorea.
MNUCSS-DMHN-01, Voucher specimens
MNUCSS-DMJJ-01) were
(MNUCSS-DMWD-01,
deposited at the College of MNUCSS-DMJH-01,
Pharmacy, Mokpo NationalMNUCSS-DMHN-01,
University (Muan, and MNUCSS-DMJJ-01)
Korea). For the present were
deposited
study, at the College
the air-dried, powdered of Pharmacy,
D. morbiferaMokpo
leavesNational
(50 g) were University
extracted (Muan, Korea).
twice with For theethanol
20–100% present
study,
(300 mL)the air-dried,
at room powdered
temperature forD. morbifera
3 days. Theleaves (50 g)was
0% extract were extracted
prepared as twice with extract
hot-water 20–100% ◦ C,
ethanol
(100
(300After
4 h). mL)filtration,
at room temperature
the resultantfor 3 days.
ethanol The 0%
solution wasextract was prepared
evaporated, as hot-water
freeze-dried, extract
and stored 50 ◦ C.
at −(100 °C,
4 h).
The After
crude filtration,
extract the resultantinethanol
was resuspended ethanolsolution
and filteredwas using
evaporated,
a 0.4-µm freeze-dried,
membrane. and stored
All the at −50
samples
°C. subjected
were The crudetoextract wasoptimization
extraction resuspendedand in ethanol
used in and filtered
the in using a 0.4-μm membrane. All the
vitro experiments.
samples were subjected to extraction optimization and used in the in vitro experiments.
3.2. Chromatographic Conditions
All analyses were performed using an Alliance 2695 HPLC system (Waters, Milford, MA,
USA) equipped with a photodiode array detector. A revese phase C18 analytical column (5-µm,
Molecules 2018, 23, 650 8 of 11

150 mm × 5 mm) was used with a mobile phase consisting of a mixture of solvent A (acetonitrile) and
B (0.2% phosphoric acid). A gradient elution (from 10/90 to 80/20 v/v) at a flow rate of 0.8 mL/min
was used and the analytical conditions are described in Table 6.

Table 6. Analytical conditions of HPLC system for analyzing two markers.

Parameters Conditions
Zorbax extended-C18
Column
(C18, 4.6 mm × 150 mm, 5 µm)
Flow rate 0.8 mL/min
Injection volumn 10 µL
UV detection 330 nm
Run time 30 min
Time (min) %A %B
0 10 90
7 10 90
Gradient 8 20 80
20 25 75
21 100 0
25 10 90
30 10 90

3.3. Method Validation

The analytical method used for the quantification of chlorogenic acid and rutin in the D. morbifera
leaf extracts was validated for specificity, linearity, sensitivity, accuracy, precision, and recovery, as
previously described [17].

3.4. Analysis of D. morbifera Leaf Extracts

The HPLC method developed inthis study was used to quantitatively determine the chlorogenic
acid and rutin contents in the extracts of D. morbifera leaves.

3.5. DPPH Free Radical Assay

The DPPH radical scavenging assay was used to evaluate the antioxidant properties of the extracts.
Briefly, various concentrations of the ethanolic extract solutions (0.5 mL) were mixed with 0.4 mM
DPPH (0.5 mL) for 10 min. The absorbance at 517 nm was measured using a microplate reader (Perkin
Elmer, Waltham, MA, USA). The radical scavenging activity was calculated as a percentage using the
following equation:

DPPH radical scavenging activity (%) = [1 − (Asample /Ablank )] × 100 (1)

IC50 (µg/mL) values were calculated from the data of the DPPH free radical scavenging activities
of the various samples [17].

3.6. Reducing Power

The reducing power assay was also used to evaluate the antioxidant properties of the extracts.
The extract (0.1 mL) was mixed with 0.2 M sodium phosphate buffer (0.5 mL), and 1% potassium
ferricyanide (0.5 mL), followed by incubation at 50 ◦ C for 20 min and the reaction was stopped by
adding 10% trichloroacetic acid solution (0.5 mL). The mixture was centrifuged at 2000× g for 10 min,
the supernatant was mixed with distilled water (0.5 mL) and 0.1% iron (III) chloride solution (0.1 mL),
and the absorbance of the final mixture was measured at 700 nm. The reducing powers of the various
samples were expressed as vitamin C equivalents [17].
Molecules 2018, 23, 650 9 of 11

3.7. Total Phenolic Content

The total phenolic content was determined using the Folin-Ciocalteu assay. An extract solution
(1 mL) or standard (gallic acid) was mixed with 1 mL each of 2% sodium carbonate and 10%
Folin-Ciocalteu phenol reagent for 10 min. The absorbance of the mixture was then measured at 750 nm
using a microplate reader (Perkin Elmer, Waltham, MA, USA). The measurement was compared to a
calibration curve constructed using gallic acid standard concentrations. The results were expressed as
milligrams of gallic acid equivalents per gram of the sample [17].

3.8. XO Inhibitory Activity In Vitro

The XO inhibitory activity was measured according to a previous report [19]. Briefly, 0.1 mL of
each sample was mixed with 0.6 mL phosphate buffer (100 mM, pH 7.4), 0.1 mL XO (0.2 U/mL), and
0.2 mL xanthine (1 mM, dissolved in 0.1 M sodium hydroxide [NaOH]) for 15 min. The reaction was
stopped by adding 0.2 mL 1 M hydrochloric acid (HCl), and the absorbance was measured at 290 nm
with ALP as the positive control.

3.9. Statistical Analysis

All data were expressed as mean ± standard deviation, and analysis of variance (post-hoc
Tukey’s multiple range test) was performed using the Statistical Package for the Social Sciences
software (version 12.0, IBM Co., Armonk, NY, USA). A p-value less than 0.05 was considered to be
statistically significant.

4. Conclusions
In the present study, hot water and ethanolic extracts of D. morbifera leaf extracts from four
different cultivation regions were successfully prepared, and their chemical profiles and biological
activities were evaluated. Hot water extracts from JH showed the highest yield and extract of the
W and K samples showed high concentrations of the selected makers. The 80% ethanolic extract
exhibited the most potent DPPH radical scavenging activity, reducing power, phenolic content, and XO
inhibitory activity. The developed HPLC method was validated and applied to identify chlorogenic
acid and rutin, which were found to be common constituents of all the D. morbifera leaf extracts.
These findings suggest that the observed antioxidant and XO inhibitory activities of D. morbifera
extracts were attributed, at least in part, to the marker compounds. To the best of our knowledge, this
is the first study to report on a validated analytical method for the standardization and optimization of
the biological properties of D. morbifera preparations. Further investigation is warranted to confirm
the in vivo pharmacological activity of D. morbifera extract and its two constituents and assess the safe
use of the plant. These propose efforts could lead to the development of D. morbifera as a potential,
effective antioxidant and anti-hyperuricemic/gout agent.

Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) grant funded
by the Korea government (MSIP; Ministry of Science, ICT & Future Planning) (No. NRF-2017R1C1B5015187).
Author Contributions: In-Soo Yoon and Seung-Sik Cho conceived and designed the experiments. Hyung-Jae Choi,
Seung-Hui Song and Seung-Sik Cho performed the experiments and analyzed the data. In-Soo Yoon and Dae-Hun
Park statistically analyzed the data.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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